The Refined Structure of Membrane Proteins Embedded in Liposomes Revealed

The role of ion channel proteins and membrane transporters is to move ions and small molecules on the cell membrane, which is very necessary for the maintenance of cell metabolism and homeostasis, but also as a variety of biological signaling pathways in the body. The researchers believe that these two types of proteins are extremely important for body health, and the defects of these proteins are often directly related to a variety of diseases; in order to understand why the defects of special proteins cause diseases, we should not only know the role of this protein, but also know how it plays a role. Further study of the structure of proteins can allow scientists to better master its mechanism of action. Since the current technology cannot perform high-resolution imaging of ion channel proteins and membrane transporters, scientists do not know the specific structure of these two proteins.

In recent years, with the help of cryo-EM (cryogenic electron microscopy) technology, scientists have begun to slowly solve the imaging problem of a variety of membrane proteins; recently, in a research report published in PNAS entitled "Cryo-EM analysis of a protein embedded in the liposome", researchers from Yan Ning's group at Princeton University used cryo-EM technology to analyze membrane proteins embedded in liposomes in detail through the study.

When X-ray crystallography is used as the mainstream research method, membrane proteins are often the most difficult targets to study in the field of structural biology. With the breakthrough of single-particle cryo-EM technology, researchers have also made some progress in elucidating the clear structure of membrane proteins. The next challenge will be how to maintain the electrochemical gradient and membrane curvature, so as to comprehensively elucidate the structure of membrane proteins, and their biological functions depend on these chemical and physical characteristics. In this study, the investigators propose a convenient workflow based on the well-characterized prototype protein AcrB for cryo-EM structural analysis of membrane proteins embedded in liposomes. After combining the optimized proteasome separation technique, preparation of low-temperature samples on graphene grids, and effective particle screening strategies, the researchers were able to obtain three-dimensional (3D) reconstructed structures of AcrB proteins embedded in liposomes at 3.9 Å resolution, and the conformation of homologous AcrB remained unchanged when the surrounding membrane showed different curvatures, and this widely used cryo-EM structural analysis method for membrane proteins with different soluble domains may lay a foundation for cryo-EM analysis of global or external membrane proteins, which will be affected by transmembrane electrochemical gradients or membrane curvatures.

The researchers say that they propose a complete workflow for high-resolution cryo-EM analysis of membrane proteins embedded in liposomes by combining optimized protein preparation steps, high-quality graphene grids, and deep 2D classification techniques. The preparation method based on exclusion chromatography (SEC) can selectively control the size of proteoliposomes, while graphene mesh can significantly improve the density and distribution of proteoliposomes in the field of view. At the same time, it also supports the reasonable data collection strategy based on low-power resolution image mode, while the deep 2D classification program can conveniently select the membrane protein particles embedded in liposomes for conventional single-particle analysis, thus avoiding the complex and highly required preprocessing.

It is worth noting that signals from soluble domains embedded in AcrB can promote particle selection in in-depth 2D classification, and it seems less practical to select transmembrane domains from membranes without this soluble domain with obvious contour/shape, therefore, the method developed by the researchers is mainly suitable for membrane proteins with obvious contour and reasonable size; on the other hand, an initial model can be used as a template to promote the screening of particles, and if a membrane protein can be successfully reconstructed into liposomes, then by conventional single-particle cryo-EM analysis, the researchers can obtain at least lower resolution 3D reconstructed images in detergent microclusters or nanodiscs, so this prerequisite does not represent a barrier to the application of this method.

Interestingly, the researchers also found that 98% of the particles in all AcrB side views possessed soluble domains in their liposomes, and localization analysis could also be extended to correct the calculation of transport efficiency in liposome functional tests, which usually have a distribution hypothesis of 50/50, which may be caused by the composition of lipids or detergents, but further understanding of liposome preferential localization requires more in-depth studies by scientists at a later stage. The researchers envisioned that the new method they developed could be applied to a series of structure-function related studies, and one of the purposes was to investigate the effects of transmembrane gradients of ions or other substrates on the function and structure of target proteins, such as voltage-gated ion channel proteins and proton-driven transporters.

At present, the biggest technical obstacle is the preparation of low-temperature samples of proteoliposomes with controllable transmembrane gradients, because the leakage of liposomes is a very serious problem, especially during sudden drop freezing; despite this, the results of this paper lay a foundation for later scientists to widely use cryo-EM technology to analyze membrane proteins and membrane structures.